This proposal will determine the structure and conformation of human factor VIII and elucidate the molecular mechanisms which result in the regulation of factor VIII activity. Factor VIII is believed to exist as a series of active heterodimers consisting of a variable length heavy chain noncovalently linked to an invariant light chain. Selected homogeneous heterodimers will be prepared and characterized in terms of native size and polypeptide composition. Detailed physico-chemical analyses will be performed on the homogeneous preparations to obtain information on conformation and intra-chain disulfide pairing. a second major focus will involve study of the structure of factor VIIIa following proteolytic activation by thrombin and will elucidate those events that result in the spontaneous inactivation of factor VIIIa. Additional studies will evaluate, both kinetically and structurally, the activation of factor VIII by factor IXa. Binding of factor VIII to factor IX (IXa) will be studied using kinetic methods, cross- linking reagents and monoclonal antibodies to determine dissociation constants, stoichiometry and location of binding sites. Sequence analysis of the transient and terminal fragments generated by proteolytic inactivation of factor VIII by activated protein C will elucidate product-precursor relationships. Comparison of rates of inactivation of factors VIII with factor VIIIa will be evaluated in terms of preferred substrates for activated protein C. These structural and mechanistic studies will offer insights into a coagulation protein that is central to the hemostatic process and will have implications for the understanding and management of hemophilia A, the most common of the congenital bleeding disorders.